A critical issue in cancer immunotherapy is raised by the finding that tumors grow in patients even when infiltrated with lymphocytes. For example, a brisk pattern of T cell infiltration into vertical growth phase melanoma is a powerful predictor of cure after surgery (Clark et al., 1989). Nevertheless, tumors frequently progress (Lee et al., 1999b). Furthermore, results from melanoma antigen-specific vaccinations suggest vaccine effectiveness in eliciting T cell responses demonstrable in circulation and at the tumor site in the absence of clinical responses (Lee et al., 1999a; Finke et al., 1999; Nielson & Maricola, 2000). An important related question is why lymphocytes that lyse tumor targets in vitro often do not demonstrate therapeutic effects after adoptive transfer to patients in vivo (Maaser et al., 1999).
Multiple factors have been suggested to underlie the absence of CTL functions in vivo, such as (1) production of TGF-β by tumor cells leading to impairment of CTL effector function, (2) downregulation of HLA class I on tumor cells in vivo leading to absence of tumor lysis by CTL, (3) absence of co-stimulatory molecules on tumor cells leading to impairment of CTL induction by direct tumor stimulation, (4) expression of Fas ligand by tumor cells leading to inactivation of Fas receptor-positive CTL, (5) emergence of tumor cell variants with epitope/antigen loss after tumor lysis by CTL in vivo, and (6) downregulation of ζ chain in CTL leading to loss of T cell function. All of these factors have been identified in vitro, but it is unclear which of them play a role in vivo. Elucidation of these questions would greatly impact T-cell based adoptive and active cancer immunotherapies.
These questions cannot be addressed directly in patients. Studies on T cell functions have been performed in tumors in situ using analyses of RNA expression (Finke et al., 1999). However, these studies have used biopsy material containing a mixture of cells and have used an RNA amplification method. Thus, the cell type which is the source of RNA (stroma cells, fibroblasts, tumor cells, immune effector cells) cannot be identified. Moreover, RNA amplification is non-linear and may be biased.
Thus far, relevant in vitro models are lacking. Traditionally, CTL are raised in two-dimensional mixed lymphocyte tumor cell culture (MLTC) that include either long-term cultured tumor cells to stimulate PBMC for CTL induction or disaggregated tumor tissue with tumor infiltrating lymphocytes (TIL), with both cultures grown directly on plastic surfaces. However, a study in melanoma patients has shown that characterization of CTL responses using tumor cell lines does not reflect the in vivo exerted anti-tumoral activity (Friedl et al., 1998). For example, melanoma-reactive human CTL derived from skin biopsies of delayed type hypersensitivity reactions, when stimulated with autologous long-term cultured tumor cells in vitro, demonstrated increased skewing of the Vβ T cell receptor (TCR) repertoire with increasing time in culture (Rao et al., 2000). This suggests the selection of a CTL population in culture that does not reflect the composition of T lymphocytes in vivo. In agreement with that study, TCR expressed by CTL raised in MLTC are not expressed in situ (Crowston et al., 1997). Changes in TCR repertoire of CTL following in vitro culture indicate changes in antigen recognition. Thus, CTL in situ may recognize different antigens than CTL derived from long-term cultures.
In addition to the alterations of T cells in MLTC upon culture, the use of long term cultured tumor cells in MLTC is disadvantageous. For example, colorectal carcinoma (CRC) cells often lose HLA class I expression in culture, whereas the same tumors express these molecules in situ (Schroder, 1995). Similarly, the tumor suppressor gene p16 is expressed in ˜70% of primary CRC, but in only ˜10% of CRC cell lines (Luger & Schwarz, 1990). Furthermore, irradiated tumor cells often used in MLTC may not produce all of the factors that non-irradiated tumor cells produce; these factors may affect the induction or effector phase of CTL.
Chemokines play a major role in inducing migration of lymphocytes, neutrophils, monocytes, macrophages and dendritic cells to the tumor site. Chemokines may be incorporated into vaccines to increase vaccine efficacy at the site of the antigen-presenting cell or attached to an anti-tumor antibody to attract the effector cells to the tumor site. Alternatively, tumors may be transduced with chemokines to attract effector cells or CTL may be transduced with chemokine receptors (Biragyn & Kwak, 2000).
Thus, there is a need in the art for model systems that can be used to identify clinically relevant behaviors of CTL, including tumor cell lysis and active migration, as well as systems that can be used to identify chemokines that influence active migration of CTL towards tumor cells.